Recombinant human thymidine phosphorylase catalyzes the reaction of arsenate with thymidine to form thymine and 2-deoxyribose 1-arsenate, which rapidly decomposes to 2-deoxyribose and inorganic arsenate. The transition-state structure of this reaction was determined using kinetic isotope effect analysis followed by computer modeling. Experimental kinetic isotope effects were determined at physiological pH and 37 degrees C. The extent of forward commitment to catalysis was determined by pulse-chase experiments to be 0.70%. The intrinsic kinetic isotope effects for [1'-(3)H]-, [2'R-(3)H]-, [2'S-(3)H]-, [4'-(3)H]-, [5'-(3)H]-, [1'-(14)C]-, and [1-(15)N]-thymidines were determined to be 0.989 +/- 0.002, 0.974 +/- 0.002, 1.036 +/- 0.002, 1.020 +/- 0.003, 1.061 +/- 0.003, 1.139 +/- 0.005, and 1.022 +/- 0.005, respectively. A computer-generated model, based on density functional electronic structure calculations, was fit to the experimental isotope effect. The structure of the transition state confirms that human thymidine phosphorylase proceeds through an S(N)2-like transition state with bond orders of 0.50 to the thymine leaving group and 0.33 to the attacking oxygen nucleophile. The reaction differs from the dissociative transition states previously reported for N-ribosyl transferases and is the first demonstration of a nucleophilic transition state for an N-ribosyl transferase. The large primary (14)C isotope effect of 1.139 can occur only in nucleophilic displacements and is the largest (14)C primary isotope effect reported for an enzymatic reaction. A transition state structure with substantial bond order to the attacking nucleophile and leaving group is confirmed by the slightly inverse 1'-(3)H isotope effect, demonstrating that the transition state is compressed by the impinging steric bulk of the nucleophile and leaving group.